Single-molecule fluorimetry and gating currents inspire an improved optical voltage indicator

Voltage-sensing domains (VSDs) underlie the movement of voltage-gated ion channels, as well as the voltage-sensitive fluorescent responses observed from a common class of genetically encoded voltage indicators (GEVIs). Despite the widespread use and potential utility of these GEVIs, the biophysical underpinnings of the relationship between VSD movement and fluorophore response remain unclear. We investigated the recently developed GEVI ArcLight, and its close variant Arclight', at both the single-molecule and macroscopic levels to better understand their characteristics and mechanisms of activity. These studies revealed a number of previously unobserved features of ArcLight's behavior, including millisecond-scale fluorescence fluctuations in single molecules as well as a previously unreported delay prior to macroscopic fluorescence onset. Finally, these mechanistic insights allowed us to improve the optical response of ArcLight to fast or repetitive pulses with the development of ArcLightning, a novel GEVI with improved kinetics. DOI: http://dx.doi.org/10.7554/eLife.10482.001

[1]  F. Bezanilla,et al.  Charge movement of a voltage-sensitive fluorescent protein. , 2009, Biophysical journal.

[2]  F. Bezanilla,et al.  S4-based voltage sensors have three major conformations , 2008, Proceedings of the National Academy of Sciences.

[3]  张静,et al.  Banana Ovate family protein MaOFP1 and MADS-box protein MuMADS1 antagonistically regulated banana fruit ripening , 2015 .

[4]  W. E. Moerner,et al.  The Fluorescence Dynamics of Single Molecules of Green Fluorescent Protein , 1999 .

[5]  Christian Eggeling,et al.  Major signal increase in fluorescence microscopy through dark-state relaxation , 2007, Nature Methods.

[6]  Vincent A. Pieribone,et al.  Single Action Potentials and Subthreshold Electrical Events Imaged in Neurons with a Fluorescent Protein Voltage Probe , 2012, Neuron.

[7]  B M Salzberg,et al.  Optical recording of voltage changes in nerve terminals and in fine neuronal processes. , 1989, Annual review of physiology.

[8]  V. Pieribone,et al.  Genetically Targeted Optical Electrophysiology in Intact Neural Circuits , 2013, Cell.

[9]  W. N. Ross,et al.  Changes in axon fluorescence during activity: Molecular probes of membrane potential , 1974, The Journal of Membrane Biology.

[10]  Dejan Zecevic,et al.  Membrane potential imaging in the nervous system , 2011 .

[11]  Walther Akemann,et al.  Engineering and Characterization of an Enhanced Fluorescent Protein Voltage Sensor , 2007, Neuroscience Research.

[12]  Yini Wang,et al.  Denaturing and refolding of protein molecules on surfaces , 2007, Proteomics.

[13]  E. Peterman,et al.  Optical methods for exploring dynamics of single copies of green fluorescent protein. , 1999, Cytometry.

[14]  Adam E. Cohen,et al.  Electrical Spiking in Escherichia coli Probed with a Fluorescent Voltage-Indicating Protein , 2011, Science.

[15]  H. Mutoh,et al.  Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain , 2014, Front. Mol. Neurosci..

[16]  B M Salzberg,et al.  A large change in axon fluorescence that provides a promising method for measuring membrane potential. , 1973, Nature: New biology.

[17]  Josef Lazar,et al.  Mechanistic Studies of the Genetically Encoded Fluorescent Protein Voltage Probe ArcLight , 2014, PloS one.

[18]  T. Gharbi,et al.  Pulsed local-field fluorescence microscopy: a new approach for measuring cellular signals in the beating heart , 2003, Pflügers Archiv.

[19]  Jin Zhong Li,et al.  Enhanced Archaerhodopsin Fluorescent Protein Voltage Indicators , 2013, PloS one.

[20]  W. Webb,et al.  Fluorescence Photoconversion Kinetics in Novel Green Fluorescent Protein pH Sensors (pHluorins) , 2004 .

[21]  H. Piao,et al.  Combinatorial Mutagenesis of the Voltage-Sensing Domain Enables the Optical Resolution of Action Potentials Firing at 60 Hz by a Genetically Encoded Fluorescent Sensor of Membrane Potential , 2015, The Journal of Neuroscience.

[22]  F. Bezanilla,et al.  Tuning the voltage-sensor motion with a single residue. , 2012, Biophysical journal.

[23]  N. Honkura,et al.  Two-photon voltage imaging using a genetically encoded voltage indicator , 2013, Scientific Reports.

[24]  D. Maclaurin,et al.  Optical recording of action potentials in mammalian neurons using a microbial rhodopsin , 2011, Nature Methods.

[25]  T. Knöpfel,et al.  Genetically encoded voltage indicators for large scale cortical imaging come of age. , 2015, Current opinion in chemical biology.

[26]  R. Tsien,et al.  On/off blinking and switching behaviour of single molecules of green fluorescent protein , 1997, Nature.

[27]  Genetic construction, properties and application of a green fluorescent protein-tagged ciliary neurotrophic factor. , 1997, Protein engineering.

[28]  Bradley J. Baker,et al.  Fluorescent Protein Voltage Probes Derived from ArcLight that Respond to Membrane Voltage Changes with Fast Kinetics , 2013, PloS one.

[29]  F. Bezanilla,et al.  Cut-open oocyte voltage-clamp technique. , 1998, Methods in enzymology.

[30]  Yasushi Okamura,et al.  Improved detection of electrical activity with a voltage probe based on a voltage‐sensing phosphatase , 2013, The Journal of physiology.

[31]  J Greve,et al.  Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Walther Akemann,et al.  Imaging neural circuit dynamics with a voltage-sensitive fluorescent protein. , 2012, Journal of neurophysiology.

[33]  Lei Jin,et al.  Genetically Encoded Protein Sensors of Membrane Potential. , 2015, Advances in experimental medicine and biology.

[34]  E. Isacoff,et al.  Genetically encoded fluorescent sensors of membrane potential , 2008, Brain cell biology.

[35]  W. Webb,et al.  Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[36]  F. Bezanilla,et al.  Properties of deactivation gating currents in Shaker channels. , 2011, Biophysical journal.

[37]  Ehud Y Isacoff,et al.  A Genetically Encoded Optical Probe of Membrane Voltage , 1997, Neuron.

[38]  G. Baldini,et al.  Protonation and conformational dynamics of GFP mutants by two-photon excitation fluorescence correlation spectroscopy. , 2008, The journal of physical chemistry. B.

[39]  Robert W. Mills,et al.  Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor , 2014, Stem cell reports.

[40]  Susumu Terakawa,et al.  Structural rearrangements in single ion channels detected optically in living cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[41]  A Watanabe,et al.  Changes in fluorescence, turbidity, and birefringence associated with nerve excitation. , 1968, Proceedings of the National Academy of Sciences of the United States of America.

[42]  T. Knöpfel,et al.  Design and characterization of a DNA‐encoded, voltage‐sensitive fluorescent protein , 2001, The European journal of neuroscience.

[43]  F. Bezanilla,et al.  Structural Implications of Fluorescence Quenching in the Shaker K+ Channel , 1998, The Journal of general physiology.

[44]  F. Bezanilla,et al.  Sensing charges of the Ciona intestinalis voltage-sensing phosphatase , 2013, The Journal of general physiology.

[45]  Paul W. Sternberg,et al.  Archaerhodopsin Variants with Enhanced Voltage Sensitive Fluorescence in Mammalian and Caenorhabditis elegans Neurons , 2014, Nature Communications.

[46]  S. Scully,et al.  Evidence for a charge-shift electrochromic mechanism in a probe of membrane potential , 1979, Nature.

[47]  Michael Z. Lin,et al.  High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor , 2014, Nature Neuroscience.

[48]  Yasushi Okamura,et al.  Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor , 2005, Nature.

[49]  V. Pieribone,et al.  A Fluorescent, Genetically-Encoded Voltage Probe Capable of Resolving Action Potentials , 2012, PloS one.

[50]  Samouil L. Farhi,et al.  All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins , 2014, Nature Methods.